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Scholars Research Library Der Pharma Chemica, 2014, 6(4):140-146 (http://derpharmachemica.com/archive.html)

ISSN 0975-413X CODEN (USA): PCHHAX

Validated UV-spectrophotometric estimation of diclazurilin API and drug product Taghreed M. Ibrahim National Organization for Drug Control and Research (NODCAR), 6-Abu Hazem Street, Pyramids Ave, Cairo, Egypt _____________________________________________________________________________________________ ABSTRACT Three simple, precise, accurate, and economical UV spectrophotometric methods have been developed and validated for the routine estimation of diclazuril in active pharmaceutical ingredient (API) and drug product. Diclazuril was estimated at 286.2 nm in UV-spectroscopic method (Method A), 260 nm in first order derivative spectroscopy (Method B) and scanned at 300 – 273 nm in Area Under Curve method (Method C).Linearity range was found to be 2 -22 µg/ml (Correlation coefficient r2 = 0.9996 in method A, r2 = 0.09997 in method B and r2 = 0.9995 in method C) in all three methods. The molar absorptivity was found to be 1.6x104 Lmol-1 cm-1 in method A, 2.038x103 Lmol-1 cm-1 in method B, and 5.5x104 Lmol-1 cm-1 in method C. These methods were tested and validated for various parameters according to ICH guidelines. The proposed methods were successfully applied for the determination of Diclazuril in drug product (powder). The results demonstrated that the procedure is accurate, precise and reproducible (% relative standard deviation < 1%), while being simple, cheap and less time consuming and can be suitably applied for the estimation of Diclazuril in powder dosage forms. Keywords: Diclazuril, UV spectrophotometric, first order derivative spectroscopy, Area Under Curve (AUC). _____________________________________________________________________________________________ INTRODUCTION Diclazuril for veterinary use, chemically is (RS) - (4-Chlorophenyl) [2, 6-dichloro-4-(3, 5-dioxo-4, 5-dihydro-1, 2, 4,-trizin-2-(3H) phenyl] acetonitrile (Fig. 1) [1, 2].Diclazuril is an antiprotozoal that has been tried in AIDS patients for the management of diarrhea associated with protozoal infection. It is used in veterinary practice for the control of coccdiosis in lambs and poultry[3], there are few methods for determination of Diclazuril in animal plasma, animal feed and eggs. In literature survey analytical methods includes liquid chromatography-tandem mass spectrometry[4, 5], gas chromatography- mass spectrometry [6] and liquid chromatography [7-9] in drug substance and drug products. But, chromatographic techniques are time consuming, costly and require expertise. This paper describes a UV-Spectrophotometric, Frist Order Derivative and UAC methods for estimation of Diclazuril. In present study, simple, economical, accurate, reproducible analytical methods with better detection range for estimation of Diclazuril in API and drug products were developed. Both these developed methods were validated as per ICH guidelines [10]. MATERIALS AND METHODS Experimental procedures: Instrument SHIMADZU UV-2450 PC Series Spectrophotometer (Tokyo – Japan) with two matched 1 cm quartz cells using the following spectral parameters: Scan mode: absorbance, Speed: fast and Slit width: 2 nm

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Taghreed M. Ibrahim Der Pharma Chemica, 2014, 6 (4):140-146 _____________________________________________________________________________ Materials Diclazuril (Batch no: 61613010)was kindly supplied by Changzhou Yabba- QH Pharmachem Colt( Ponds Co.) and assayed for purity according to the official potentiometric titration[1,2 ]to contain 99.80%±0.19%.Primazuril 0.2% powder B.N. 13015, each one gram was labeled to contain 2.0mg of Diclazurilmanufactured by Pharm Cure Pharmaceutical Industries for PRIMA VET.Curacoxin 0.5% B. N. 2014/1, each one gram contain 5.0 mg of Diclazuril manufactured by Arabcomed Co. N, N Dimethylformamide, laboratory reagent grade, FissionsScientific Equipment, Bishop Meadow, Southborough, LE 11 ORG, England, Code: D/3840/15 Lot: 1127285.

Fig.1: Diclazuril

Analytical method development Different solvents were investigated to develop suitable UV-spectrophotometric methods for the analysis of Diclazuril dosage form. Diclazuril practically insoluble in water, alcohol, and methylene chloride, sparingly soluble in dimethylformamide. For the selection of solvent the critical employed were sensitivity of the method, ease of sample preparation, solubility of the drug, cost of the solvent and applicability of the method to various purposes. Absorbance of the Diclazuril in selected solvent at respective wavelength was determined and molar absorptivity and Sandal's sensitivity was calculated according to the standard formulae (Table 1).

Fig.2: zero- order spectrum of 10 µg/ml diclazuril at ʎmax286.2 nm in dimethylformamide

Calibration standards Accurately about 100 mg of the drug substance was weighed and dissolved in dimethylformamide and the volume is made up to the volume 100 ml to give standard stock solution (1000µg/ml) and from this solution 10 ml of sample was transferred in to separated 100 ml volumetric flask and volume was made up to the mark 100 ml with dimethylformamide to get concentration 100µg/ml as a second stock. Aliquots of standard stock solution were

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Taghreed M. Ibrahim Der Pharma Chemica, 2014, 6 (4):140-146 _____________________________________________________________________________ pipetted out and suitably with dimethylformamide to get the final concentration of 2,4,6,8,10,12,14,16,18,20, and 22 µg/ml of standard stock solution. UV-Spectrophotometric method (Method A) For the selection of analytical wavelength, 10µg/ml working standard solution was prepared by appropriate dilution of standard stock solution and blank solution in dimethylformamide was used. This solution was scanned in the spectrum mode from 400 nm to 200 nm. From the spectra of drug (Fig. 2) ʎmax of Diclazuril 286.2 nm was selected for the analysis. A calibration curve was plotted by taken the absorbance against the concentration of standard stock solutions (Graph 1). By using the calibration curve, the concentration of sample solution can be determined. 1 0.122x + 0.039y = 0.999= ² R

0.9 0.8 Absorbance

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

2

4

6

8

10 12 14 16 18 20 22 24

Concentration ug/ml Graph 1: UV calibration curve of diclazuril

First order derivative spectroscopy (Method B) In this method, 10µg/ml working standard solution was prepared by appropriate dilution of standard stock solution in dimethylformamide and it used as blank solution. This solution was scanned from 400nm to 200nm wavelength ranges and the first order derivative spectra were obtained at n= 1, a sharp peak at 260 nm (Fig.3). The absorbance difference at n=1 (dA /dʎ) was calculated, which is directly proportional to the concentration of the standard stock solution. A calibration curve was plotted by taking the absorbance difference (dA /dʎ) against the concentration of standard stock solutions (Graph 2). By using the calibration curve, the concentration of the sample solution can be determined.

Fig.3: first derivative spectrum of 10 µg/ml diclazuril showing amplitude at 260 nm in dimethylformamide

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Taghreed M. Ibrahim Der Pharma Chemica, 2014, 6 (4):140-146 _____________________________________________________________________________ 0.14 0.002x - 0.005y = 0.999= ² R

0.12

dA / dʎ

0.1 0.08 0.06 0.04 0.02 0 0

2

4

6

8

10 12 14 16 18 20 22 24

Concentration ug/ml Graph 2: Frist derivative calibration curve of diclazuril

Area Under Curve method (Method C) This method is applicable when there is no sharp or when broad spectra obtained. It involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths ʎ1 and ʎ2. Area calculation processing item calculates the area bound by the curve and the horizontal axis. The horizontal axis is selected by entering wavelength range over which the area has to be calculated. This wavelength range is selected on the basis of repeated observation so as to get the linearity between area under curve [11] and concentration. For the selection of analytical wavelength, 10µg/ml working standard solution was prepared by appropriate dilution of standard stock solution in dimethylformamide and it used as blank solution. This solution was scanned in the spectrum mode from 400nm to 200nm (Fig.4). From the spectra of drug, area under curve in the range of 300 – 273nm was selected for the analysis. The calibration curve was plotted as AUC against concentration of standard stock solution (Graph 3). By using the calibration curve, the concentration of the sample solution can be determined.

Fig.4: Area under curve spectrum of 10 µg/ml diclazuril showing at 300nm – 273nm in dimethylformamide

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Taghreed M. Ibrahim Der Pharma Chemica, 2014, 6 (4):140-146 _____________________________________________________________________________ 3.5 3

0.247x + 0.134y = 0.999= ² R

Absorbance

2.5 2 1.5 1 0.5 0 0

2

4

6

8

10 12 14 16 18 20 22 24

Concentration ug/ml Graph 3: Area under curve calibration curve of diclazuril

Analytical Validations Specificity and Selectivity Diclazuril solutions (10µg/ml) were prepared in dimethylformamide along with and without common excipients [aerosol and lactose monohydrate].All the solutions were scanned in the range 400-200 nm and checked for in absorbance at respective wavelength. In a separate study, drug concentration of 10µg/ml was prepared independently from pure drug stock and commercial sample stock in selected media and analyzed (N=6) t-test at 95% level was performed to compare the means absorbance (Table 1). Table 1: Optical characteristics, statistical data of the correlation and validation parameters for Diclazuril by UV spectroscopy, first order derivative & AUC method Parameters Method A Method B Method C ʎ max 286.2 nm 260.0 nm 300.0 – 273.0 nm -1 -1 4 3 Molar absorptivity (L mol cm ) 1.6x10 2.038x 10 5.5x104 -2 -3 -3 Sandal sensitivity ( µcm /0.001 abs unit ) 2.20x10 1.83x10 6.90x10-3 Regression equation (Y= a+ bc) 0.005 0.135 Slope (b) 0.039 0.000478 0.00447 0.00234 SD of slope 0.00153 0.000368 0.00436 S E of slope Confidence limit of slope 0.038087 – 0.040128 0.005492 – 0.005671 0.1300 01 – 0.13849 0.247 Intercept (a) 0.122 -0.0027 0.000623 0.08100 SD of Intercept 0.0004821 0.02133 0.000603 S E of Intercept 0.005075 -0.00444 - -0.00109 0.18849 – 0.30584 Confidence limit of Intercept 0.1084 – 0.136579 0.9995 0.9996 0.9997 Correlation coefficient (r ) 0.00073 0.02561 0.00615 SE of ( r) Validation parameters Selectivity and Specificity 0.175 1.15 0.491 (t-test) (2.228) Accuracy(Mean ± RSD %) 99.845 ± 0.867 % 99.0 ± 0.957 % 99.916 ± 0.862% Linearity (µg /ml) 2.0 -22.0 µg /ml 2.0 -22.0 µg /ml 2.0 -22.0 µg /ml Limit of detection [LOD] 0.825 µg /ml 0.891 µg /ml 0.726 µg /ml Limit of quantitation [LOQ] 2.500 µg /ml 2.700 µg /ml 2.200 µg /ml Robustness(mean% recovery ±S.D) 100.11% ± 0.545 100.08% ± 0.416 100.4% ± 0.583 Y = bC + a where C is the concentration of Dicalzuril in µg /ml and Y is absorbance unit. (Each value is a result of six separate determinations), c 95% confidence limit.

Precision Repeatability was determined by using different levels of drug concentrations (same concentration levels taken in accuracy study), prepared from independent stock solution and analyzed (N=6). Inter-day and intra-day variation and instrument variation were taken to determine intermediate precision of the proposed methods (N=6). The % relative standard deviation of the predicted concentrations from the regression equation was taken as precision (Table 2).

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Taghreed M. Ibrahim Der Pharma Chemica, 2014, 6 (4):140-146 _____________________________________________________________________________ Table 2: results of intermediate precision study Concentration (in µg / ml) Method A 2 12 22 Method B 2 12 22 Method C 2 12 22

Inter-day repeatability % RSD (N = 6) Day 1 Day 2 Day 3

Intra-day repeatability(N = 6) Mean ±% RSD

Inter-instrument* Repeatability(N = 6) Mean ± % RSD

0.1783 0.5992 0.9778

0.1566 0.3214 0.7210

0.1134 0.3188 0.5379

100.11± 0.439% 100.08 ± 0.306 % 100.23 ± 0.601%

100.06 ± 0.653 100.08 ± 0.257 100.04 ± 0.206

0.009 0.064 0.120

0.005 0.051 0.110

0.001 0.035 0.009

100.21 ± 0.619% 100.16 ± 0.421% 100.44 ± 0.472%

100.10± 0.276% 99.98 ± 0.383% 99.99 ± 0.299%

0.500 0.328 0.155 100.00± 0.228% 1.850 1.365 0.998 100.15 ± 0.507% 3.184 2.367 1.581 100.08 ± 0.285% *Instrument 1:SHIMADZU UV-2450 PC Series Spectrophotometer Instrument 2: UNICAM UV 300 Spectrophotometer

100.14 ± 0.229% 100.12 ± 0.310% 100.20± 0.227%

Accuracy As a part of determining accuracy of the proposed methods, different levels of drug concentrations were prepared from independent stock solution and analyzed (N=6). Accuracy was assessed as the standard deviation and percentage relative standard deviation studies were found to be satisfactory (Table 1). To give additional support to accuracy of the developed assay method, standard addition method was done. The percent recovery of the added API was calculated as, % Recovery = [(Cy- Cu) / Ca ] Χ 100 Where Cy is the total drug concentration measured after standard addition, Cu is the drug concentration in formulation and Ca is the drug concentration added to the formulation (Table 3). Table 3: results of standard addition method

Method

Method A

Method B

Method C

Concentration of drug in formulation (µg /ml) 10 10 10 10 10 10 10 10 10

Concentration of pure drug added (µg /ml) 2 12 20 2 12 20 2 12 20

% level of pure drug added 20 120 200 20 120 200 20 120 200

Total concentration of drug found (µg /ml) 11.98 22.12 30.24 12.02 21.90 29.88 12.10 22.21 30.11

% Analytical recovery ± SD 99.77±0.01 101.21±0.14 102.05±0.01 100.23±0.01 99.80±0.53 99.00±0.07 100.25±0.12 101.10±0.10 102.00±0.04

Linearity To establish linearity of the proposed method, six separate series of solutions of the drug (2 -22 µg/ml) were prepared from the stock solution and analyzed. Limit Of Detection (LOD) and Limit Of Quantitation (LOQ) The LOD and LOQ of the Diclazuril by proposed methods were determined by using calibration standard. LOD and LOQ were calculated as 3.3σ /S and 10σ /S, respectively, where S is slope of the calibration curve and σ is standard deviation of y-intercept of regression equation( Table 1). Robustness Robustness of the proposed method was determined by stability of the Diclazuril at room temperature for 8 hours. The different concentrations were prepared in dimethylformamide.Mean percentage recovery was determined(Table 1). ESTIMATION OF DICLAZURIL FROM DRUG PRODUCT The validity of the suggested methods was assessed by applying the standard addition technique by adding Diclazuril to the previously analyzed drug products. Statistical comparison of the results obtained by the proposed method with those obtained by HPLC reported method [12-13] showed that the recommended procedures are simpler and sensitive without any loss of accuracy or precision (Table 3, 4).

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Taghreed M. Ibrahim Der Pharma Chemica, 2014, 6 (4):140-146 _____________________________________________________________________________ Table 4: Analysis of Diclazuril in its drug products by the proposed (method A, method B, and method C) methods and compared with the reported HPLC method Parameter Mean* ± RSD% Variance SE t-test (2.228)a F-test (5.1)a

Method A 97.2 0.551 0.302 0.224 1.23 2.00

Primazuril 0.2% powder Curacoxin 0.5% powder Method B Method C Reported HPLC[12] Method A Method B Method C Reported HPLC[13] 96.7 95.8 94.5 99.43 99.23 99.05 98.82 0.611 0.264 0.388 1.11 0.48 0.521 0.609 0.373 0.069 0.151 1.23 0.23 0.272 0.372 0.249 0.107 0.092 0.453 0.272 0.212 0.248 1.56 1.99 2.1 1.91 1.23 2.47 2.19 3.3 1.61 1.36 * Average of six different experiments. aTheoretical values.

RESULTS AND DISCUSSION The methods discussed in the present work provide a convenient and accurate way for the analysis of Diclazuril in its drug product. Absorbance maxima of Diclazuril at 286.2 nm (Method A) and in the first order derivative spectra, sharp peak in 260 nm (Method B) were selected for the analysis. Method C was area under curve (AUC) and the wavelength range for quantitation was 300 – 273 nm. Linearity for detector response was observed in the concentration range of 2 -22 µg/ml for all three methods.Percentage label claim for Diclazuril in powder analysis, by all the three methods, was found to be in the range of 95.80% to 99.43%. Standard deviation and correlation coefficient for powder, by all the methods, was found to be less than ± 1.0 indicating the precision of the methods. Accuracy of proposed methods was ascertained by recovery studies and the results are expressed as % recovery. Percentage analytical recovery for Diclazuril, by all the methods, was found in range of 99.00% to 102.05% values of standard. CONCLUSION In summary, the proposed methods were less time consuming, rapid, accurate, precise and inexpensive and can be used for routine analysis of Diclazuril in API and drug products. The sample recoveries in powder and oral suspension were in good agreement with their respective label claims and suggested non-interference of excipients in the estimation of drug products. REFERENCES [1] British Pharmacopoeia Veterinary, The Stationary Office Medicinal and Pharmaceutical Substance, London, 2014, Vet.59-60. [2] European Pharmacopeia- 8th Edition, 2014, 2034-2035. [3] Sean C Sweet man, BParm, FRPharmS, Martindale The Complete Drug Reference, Thirty- seventh edition, The Stationery Office on behalf of the Medicine and Healthcare products Regulatory Agency (MHRA), 918. [4] L. Mortimer, E.Daeseleire, P.Delahaut, J. Anal. Chime. Act, 2003, 483(1-2), 27-37. [5]S. Corbels,M.Cher let,P. de-Backer,J. Rapid, Common, Mass, Spectrum, 2002, 16(15), 1463-1469. [6]W.Blanch flower,P. Hughes,A.Canova,D. Kennedy,J. AOAC Int., 1994, 77(5), 1061-1065. [7]Q. Shao,J. Zhang,S.Xian.Chou,H. Chen,Q. Sheen, X. ZR,J. Fenxi,Huaxue 2003; 31(11):1376-1379. [8]A. Fontaine, K. Haustraete,J. AOAC-Int. 1994, 77(6), 1359-1361. [9]J. Kick,M.Smut,R.Sneers,J.Chromatogram. 1992, 606(1), 141-146. [10] ICH Q2RI 2005 Validation of Analytical Procedures Text and Methodology, in: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. [11] A.Sandpit, K.Lathe, Journal Pharm Educ.Res.2007; 41(4): 353-7. [12] Personal communication; Pharma cure pharmaceutical Industries for PRIMA VET. (2009), specification. Cairo, Egypt. [13] Personal communication; ARABCOMED pharmaceutical Industries VET. (2009), specification. Cairo, Egypt.

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